This invention relates to a bioactive extract or concentrate prepared from basement membranes of natural tissues and the use of those extracts to promote cell growth and proliferation. More particularly, this invention is directed to submucosal tissue extracts and their preparation and use in promoting cell growth in vitro and in vivo.
It has been reported that various basement membranes and other extracellular matrices can be utilized as tissue graft constructs or as cell culture substrates. Several matrix products derived from complex substrates are commercially available for use in supporting cell growth in vitro. For example, Becton Dickinson currently offers two such products: Human Extracellular Matrix and MATRIGEL(copyright) Basement Membrane Matrix. Human Extracellular Matrix is a chromatographically partially purified matrix extract derived from human placenta and comprises laminin, collagen IV, and heparin sulfate proteoglycan. (Kleinman, HK et al., U.S. Pat. No. 4,829,000 (1989)) MATRIGEL(copyright) is a soluble basement membrane extract of the Engelbreth-Holm-Swarm (EHS) tumor, gelled to form a reconstituted basement membrane. Both of these matrix products require costly biochemical isolation, purification, and synthesis techniques and thus production costs are high.
Submucosal tissue harvested from warm blooded vertebrates is a collagenous matrix that has shown great promise as a unique graft material for inducing the repair of damaged or diseased tissues in vivo, and for inducing the proliferation and differentiation of cell populations in vitro. Submucosal tissue consists primarily of extracellular matrix material prepared by mechanically removing selected portions of the mucosa and the external muscle layers and then by lysing resident cells with hypotonic washes. Preliminary biochemical analyses show that the composition of small intestinal submucosa is similar to that of other basement membrane/extracellular matrix structures, and consists of a complex array of collagens, proteoglycans, glycosaminoglycans, and glycoproteins. The major components commonly identified in extracellular matrix tissues similar to submucosal tissue include the cell adhesion proteins, fibronectin, vitronectin, thrombospondin, and laminin; the structural components, collagens and elastin; and the proteoglycans, serglycin, versican, decorin, and perlecan.
Numerous studies have shown that submucosal tissue is capable of inducing host tissue proliferation, remodeling and regeneration of tissue structures following implantation in a number of in vivo microenvironments including lower urinary tract, body wall, tendon, ligament, bone, cardiovascular tissues and the central nervous system. Upon implantation, cellular infiltration and a rapid neovascularization are observed and the submucosa extracellular matrix material is remodeled into host replacement tissue with site-specific structural and functional properties.
Submucosal tissue can be obtained from various sources, including intestinal tissue harvested from animals raised for meat production, including, for example, pigs, cattle and sheep or other warm-blooded vertebrates. As a tissue graft, submucosal tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. It has been used successfully in vascular grafts, urinary bladder and hernia repair, replacement and repair of tendons and ligaments, and dermal grafts. The preparation and use of submucosa as a tissue graft composition is described in U.S. Pat. Nos. 4,902,508; 5,281,422; 5,275,826; 5,554,389 and other related U.S. patents.
Direct interactions between extracellular matrix components and cells are known to mediate processes which are fundamental to migration, proliferation and differentiation during development. However, the role of the extracellular matrix in wound healing and tissue regeneration has been less well studied. It has been found in accordance with the present invention that submucosal tissue can be extracted to provide compositions comprising isolated bioactive components of the submucosal tissue in their active form. Such an enriched extract can be utilized as an additive for tissue culture media to promote in vitro cell growth and proliferation, and also can be used as an active ingredient for wound healing compositions such as topically applied creams and bandages.